| Rice(Oryza saliva L.)is one of the most important crop which plays an important role in food security.Heading date is one of the most important agronomic traits of rice.Study on heading date could provide theoretical and material foundation for wide adaptability and high-yield rice breeding.Related studies show that the process flowering formation in rice is affected by various environmental and endogenous genetic network,but their accurate molecular regulation mechanism are still need to elucidated.In our study,two stable heredity early heading mutants,em148 and em61,was isolated from the ethyl methanesulfonate(EMS)mutagenesis library of japonica rice mutant lvpl of Nipponbare.Single gene mutant of EM148 was constructed and named e148,meanwhile,single gene mutant of EM61 was constructed and named e61.Then,we surveyed and compared the phenotype and major agronomic traits of the mutants and their controls,and made genetic analysis and MutMap cloning.The main results were described as follows.1)Comparing the heading date and the main agronomic characters of em61 and e61,both of them showed a earlier heading phenotypic than their controls,28 d and 20 d,respectively.Genetic analysis indicated that em148 was controlled by a single recessive nuclear gene.Using MutMap method,the re-sequencing data of the mutant DNA pool was aligned with the Nipponbare reference genome.Accroding to the difference SNP and its linkage with the mutant gene,we located the gene in 10M-13M on chromosome 3 short-arm.No reported rice heading date genes were found in this interval,suggesting that the mutant gene EM148 is a new rice heading gene.Screening and sequencing of candidate genes in this area,only one transcription factor gene EM148 was found mutated,a single base guanine Thymine(T)was substituted by a Cytosine(C)at the CDS sequence 626th position,which is a missense mutation,made the encoded amino acids Ser substituted by Gly.Then,functional complementarity,RNA interference and CRISPR knockout transgenic validation were used the to verify the candidate gene.Simultaneously,the expression pattern of the gene was studied.It was found that the expression level of the gene in the reproductive growth stage was significantly higher than that of the vegetative growth stage,suggesting that it was important in the flowering transformation.2)Comparing the heading date and the main agronomic characters of em61 and e61,both of them showed earlier heading than the controls,Respectively,early 23 d and 15 d.In the previous study,we have completed cloning the gene of em61 and showed that EM61 was probably to be the allele of Se14.Furthermore,expression of heading date genes such as SDG724,Se14,Ehd1,Hd3a and RFT1 were analyzed using Quantitative RT-PCR in em61,e61 and their controls lvp1 and NP,the results showed that EM61 regulates the process of heading of rice by affecting the expression of Eh1,Hd3a and RFT1,thus suppressed rice heading in long day condition.In the double mutant,EM61 could partially respond to the expression level of the related genes in the heading stage of lvpl,leading to the early heading of em61 flowering stage,partially recovering the heading date of lvp1.In addition,em61 and e61 also showed a slightly shorter plant height,smaller leaf angle,shorter internode length of panicle and so on,the BR related genes were analyzed using Quantitative RT-PCR,the results showed that the expression of BR pathway-related genes such as D11,D2,BU1 and BRI1 was significantly decreased in em61 and e61 compared to their controls.It is suggested that mutant gene EM61 could affect the expression of BR pathway-related genes,thus affecting the process of rice grocontrolh and development. |
